1. Field of the Invention
The present invention pertains to the art of measuring the physical configuration of a molecule and, more particularly, to sequencing individual monomers of a polymer via measuring the change in ionic current that is produced by the polymer when it passes through a narrow constriction within a fluid channel.
2. Discussion of the Prior Art
There has been considerable interest in recent years in measuring the interaction kinetics of single molecules with ion channels and associated protein pores by the method of recording the momentary change in the ionic current that passes through the channel while the molecule is present. In general, the molecule partially blocks the current flow with an amplitude and duration that are characteristic of the particular physical size and configuration of the molecule, and its chemical interaction with active sites within the channel. Efforts are underway to apply this current blocking method as the basis of a sensor system that identifies the presence of individual target molecules within a background of typical environmental chemical species.
A particular application of the current blocking method is to identify individual monomers within a polymer and, in the ideal case, is sequence the individual bases of complex biological polymers such as DNA and RNA. One of the most promising methods being investigated under the Human Genome Project is to drive single stranded DNA (ssDNA) through a nanoscale channel and attempt to measure the characteristic current change through the channel for each nucleotide base. These methods have been implemented using either a naturally occurring pore, such as alpha hemolysin (aHl), suspended in a bilayer, or using nanometer scale pores in a substrate such as silicon. However, advancements with both designs have been unable to successfully, rapidly sequence DNA because the DNA translocates through the pore too quickly for the electronics to measure the rapid and small changes in current produced by each nucleotide base.
Translocation rates for single stranded polymers under 120 millivolt (mV) applied voltage at 25° C. are on the order of 100 micrometers per second (μm/sec) to 500 μm/sec, providing an average time to measure the signal corresponding to an individual monomer of DNA or RNA (i.e. localize to the spatial extent to a single base) in the order of 0.8 to 4 microseconds (μs) per base. The electric field force on the DNA is balanced by an average force produced by hydrodynamic drag and the molecular interaction with the channel, resulting in a translocation velocity that is proportional to the applied force. Thus, if the applied voltage that produces the electric field is reduced from 100 mV to 10 mV, the velocity of the polymer is reduced ten fold, giving times ranging from 8 μs to 40 μs per base. Reducing the applied voltage thereby offers a means to increase the time a given DNA base is located in the appropriate region of the pore. Increasing the available time allows a number of measurements to be made and averaged in order to improve the accuracy of the measurement. However, in conventional systems, the applied voltage also produces the current that gives the signal characteristic of the particular blocking event. This creates the conflict that if the applied voltage is reduced the current is reduced in direct proportion. Conversely, if the applied voltage is increased to produce a larger current, the DNA translocates faster, giving less time to measure the signal.
Ideally, the molecular translocation rate is reduced to a very low level to allow measurement times on the order of 1 millisecond (ms) per base. However, as the molecule translocates more slowly, the relative effect of thermally induced (Brownian) motion on the molecule will increase. The net distance traversed due to Brownian motion scales as the square root of the total time interval. Thus, a molecule that resides at a nominal point for 1 ms will move an average net distance away from that point due to thermal motion that is 32 times further than if it resided for 1 μs. In the case of sequencing DNA, random thermally induced motion sets a limit to how much the measurement time can be practically increased by merely slowing down the translocation rate of the DNA: attempts to measure a single monomer over long time periods allow random thermally induced motion to blur the results by mixing in contributions from neighboring monomers.
Methods developed that attempt to slow down the rate of DNA translocation include the use of hairpins captured in the entry of the channel that stop DNA translocation and unzip slowly, as well as polymerases and exonucleases that act as molecular machines to draw the DNA through the channel base-by-base during the enzyme cycle. In both cases, diffusional motion of DNA is reduced by the tension within the DNA caused by the electric field force pulling against the translocation limiting mechanism. However, the DNA hairpins can only be used as short strands of DNA, and the enzyme methods have not yet advanced to the point that they can control the translocation rate of a molecule.
Recently, variations in the hairpin approach have allowed the detection of distinct, individual blocking signals for all four DNA bases, with signal differences in the order of ˜1 picoamp (pA) to 4 pA (at 120 mV bias). In addition, detection of a single base change within a polybase chain has been shown, indicating that only a single base is needed for measurement. Based on these measured signal amplitudes, present ionic current recording technology is in the order of 100 times too insensitive at the detection bandwidths (short measurement durations) that are required.
Accordingly, what is needed is a system and method to reduce the translocation velocity of a molecule through a detection channel and the diffusional motion of the molecule, while translocating, that is not limited to short molecular lengths and, in addition, does not inherently act to reduce the current blocking signal of interest.